Isotopically enriched N-substituted piperazines and methods for the preparation thereof

ABSTRACT

In some embodiments, this invention pertains to isotopically enriched N-substituted piperazines. In some embodiments, this invention pertains to methods for the preparation of isotopically enriched N-substituted piperazines.

CROSS REFERENCE TO RELATED APPLICATIONS Field of the Invention

In some embodiments, this invention pertains to isotopically enrichedN-substituted piperazines. In some embodiments, this invention pertainsto methods for the preparation of isotopically enriched N-substitutedpiperazines.

INTRODUCTION

In some embodiments, this invention pertains to isotopically enrichedN-substituted piperazines. In some embodiments, this invention pertainsto methods for the preparation of isotopically enriched N-substitutedpiperazines. N-substituted piperazines can be used as intermediates inthe synthesis of N-substituted piperazine acetic acids. N-substitutedpiperazine acetic acids can be used as intermediates in the synthesis ofactive esters of N-substituted piperazine acetic acid. Active esters arewell known in peptide synthesis and refer to certain esters that areeasily reacted with an amine of an amino acid under conditions commonlyused in peptide synthesis (For a discussion of active esters please see:Innovation And Perspectives In Solid Phase Synthesis, Editor: RogerEpton, SPCC (UK) Ltd, Birmingham, 1990).

The active esters of N-substituted piperazine acetic acid can be used aslabeling reagents. In some embodiments, a set of isobaric labelingreagents can be prepared. The set of isobaric labeling reagents can beused to label analytes, such as peptides, proteins, amino acids,oligonucleotides, DNA, RNA, lipids, carbohydrates, steroids, smallmolecules and the like. The labeled analytes can be mixed together andanalyzed simultaneously in a mass spectrometer. Because the heavy atomisotope distribution in each of the isobaric labeling reagents can bedesigned to result in the generation of a unique “signature ion” whenanalyzed in a mass spectrometer (MS), labeled components of the mixtureassociated with each of the labeling reagents, and by implicationcomponents of each labeling reaction used to produce the mixture, can bedeconvoluted. Deconvolution can include determining the relative and/orabsolute amount of one or more labeled components in each of theindividual samples that were labeled and combined to form the mixture.The N-substituted piperazine acetic acid active esters described hereintherefore can be powerful tools for analyte analysis, including but notlimited to multiplex proteomic analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a synthetic scheme for the synthesis ofN-methyl piperazines.

FIG. 2A is an illustration of a synthetic scheme for the synthesis ofN-methyl piperazine acetic acids.

FIG. 2B is an illustration of another synthetic scheme for the synthesisof N-methyl piperazine acetic acids.

FIG. 2C is an illustration of yet another synthetic scheme for thesynthesis of N-methyl piperazine acetic acids.

FIG. 3A is an illustration of a synthetic scheme for the synthesis of¹⁸O labeled N-methyl piperazine acetic acids.

FIG. 3B is an illustration of another synthetic scheme for the synthesisof ¹⁸O labeled N-methyl piperazine acetic acids.

FIG. 4A is an illustration of a synthetic scheme for the synthesis ofvarious active esters of N-methyl piperazine acetic acid.

FIG. 4B is an illustration of another synthetic scheme for the synthesisof various active esters of N-methyl piperazine acetic acid.

FIG. 4C is an illustration of yet another synthetic scheme for thesynthesis of various active esters of N-methyl piperazine acetic acid.

FIG. 4D is an illustration of still another synthetic scheme for thesynthesis of various active esters of N-methyl piperazine acetic acid.

FIG. 5A is an illustration of the heavy atom isotope incorporationpathway for the preparation of four isobaric N-methyl piperazine aceticacids.

FIG. 5B is an illustration of the labeling and fragmentation of peptidesusing four isobaric N-methyl piperazine acetic acid active esterlabeling reagents.

DEFINITIONS

For the purposes of interpreting of this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa:

As used herein, “analyte” refers to a molecule of interest that may bedetermined. Non-limiting examples of analytes include, but are notlimited to, proteins, peptides, nucleic acids (both DNA or RNA),carbohydrates, lipids, steroids and other small molecules with amolecular weight of less than 1500 Daltons (Da). The source of theanalyte, or the sample comprising the analyte, is not a limitation as itcan come from any source. The analyte or analytes can be natural orsynthetic. Non-limiting examples of sources for the analyte, or thesample comprising the analyte, include cells or tissues, or cultures (orsubcultures) thereof. Non-limiting examples of analyte sources include,but are not limited to, crude or processed cell lysates, body fluids,tissue extracts, cell extracts or fractions (or portions) from aseparations process such as a chromatographic separation, a 1Delectrophoretic separation, a 2D electrophoretic separation or acapillary electrophoretic separation. Body fluids include, but are notlimited to, blood, urine, feces, spinal fluid, cerebral fluid, amnioticfluid, lymph fluid or a fluid from a glandular secretion. By processedcell lysate we mean that the cell lysate is treated, in addition to thetreatments needed to lyse the cell, to thereby perform additionalprocessing of the collected material. For example, the sample can be acell lysate comprising one or more analytes that are peptides formed bytreatment of the cell lysate with a proteolytic enzyme to thereby digestprecursor peptides and/or proteins.

Except as when clearly not intended based upon the context in which itis being used (e.g. when made in reference to a structure that dictatesotherwise), “ester” refers to both an ester and/or a thioester.

As used herein, “fragmentation” refers to the breaking of a covalentbond.

As used herein, “fragment” refers to a product of fragmentation (noun)or the operation of causing fragmentation (verb).

As used herein, “isotopically enriched” means that a compound (e.g.labeling reagent) has been enriched synthetically with one or more heavyatom isotopes (e.g. stable isotopes such as Deuterium, ¹³C, ¹⁵N, ¹⁸O,³⁷Cl or ⁸¹Br). Because isotopic enrichment is not 100% effect can beimpurities of the compound that are of lesser states of enrichment andthese will have a lower mass. Likewise, because of over-enrichment(undesired enrichment) and because of natural isotopic abundance, therecan be impurities of greater mass.

As used herein, “labeling reagent” refers to a moiety suitable to markan analyte for determination. The term label is synonymous with theterms tag and mark and other equivalent terms and phrases. For example,a labeled analyte can be referred to as a tagged analyte or a markedanalyte.

As used herein, “natural isotopic abundance” refers to the level (ordistribution) of one or more isotopes found in a compound based upon thenatural prevalence of an isotope or isotopes in nature. For example, anatural compound obtained from living plant matter will typicallycontain about 0.6% ¹³C.

Description of Various Embodiments of the Invention

I. Preparation of N-Substituted Piperazines Comprising Heavy AtomIsotopes

In some embodiments, this invention pertains to a method for theproduction of isotopically enriched N-substituted piperazines, and theN-substituted piperazines themselves. According to the method, apartially protected amino acid can be condensed with an N-substitutedamino acid ester wherein at least one of the two amino acids comprises aheavy atom isotope such as, for example, ¹⁸O, ¹⁵N, ¹³C, ⁸¹Br, ³⁷Cl ordeuterium. When condensing the two amino acids, any side chain reactivegroups can be protected as they would be for the condensation of aminoacids to form peptides. Similarly, the condensation chemistry can bechosen from the various methods known for condensing amino acids. Theseinclude, but are not limited to, the use of carbodiimides (e.g.dicyclohexylcarbodiimide, DCC), active esters, mixed anhydride formationand the like.

The partially protected amino acid comprises an amine-protecting group(N-protecting group), such as tert-butyloxycarbonyl (t-boc); awell-known protecting group in peptide synthesis. The partially protectamino acid can comprise a side chain protecting where the amino acidcomprises a reactive side chain moiety. The amino acid can be anynatural amino acid (e.g. glycine, alanine, lysine) or non-natural aminoacid of basic structure:

wherein Pg can be the N-protecting group. Each group Z can beindependently hydrogen, deuterium, fluorine, chlorine, bromine, iodine,an amino acid side chain, a straight chain or branched C1-C6 alkyl groupthat may optionally contain a substituted or unsubstituted aryl groupwherein the carbon atoms of the alkyl and aryl groups each independentlycomprise linked hydrogen, deuterium or fluorine atoms, a straight chainor branched C1-C6 alkyl ether group that may optionally contain asubstituted or unsubstituted aryl group wherein the carbon atoms of thealkyl and aryl groups each independently comprise linked hydrogen,deuterium or fluorine atoms or a straight chain or branched C1-C6 alkoxygroup that may optionally contain a substituted or unsubstituted arylgroup wherein the carbon atoms of the alkyl and aryl groups eachindependently comprise linked hydrogen, deuterium or fluorine atoms. Insome embodiments, each Z is independently hydrogen, methyl or methoxy.In some embodiments, each Z is hydrogen, deuterium, fluorine, chlorine,bromine or iodine. An alkyl ether group, as used herein, can include oneor more polyethylene glycol substituents. Similarly, the alkoxy group,as used herein, can comprise ether and/or polyethylene glycolsubstituents. The N-protecting group can be an acid labile protectinggroup. The N-protecting group can be a base labile protecting group.

The N-substituted amino acid ester can be any natural amino acid (e.g.glycine, alanine, lysine) or non-natural amino acid of basic structure:

wherein Z is previously defined above. The group Y can be a straightchain or branched C1-C6 alkyl group or a straight chain or branchedC1-C6 alkyl ether group wherein the carbon atoms of the alkyl group oralkyl ether group each independently comprise linked hydrogen, deuteriumor fluorine atoms. In some embodiments, Y is methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl. The group R canbe a straight chain or branched C1-C6 alkyl group or a substituted orunsubstituted phenyl group, wherein the carbon atoms of the alkyl groupor phenyl group each independently comprise linked hydrogen, deuteriumor fluorine atoms. In some embodiments, the N-substituted amino acidester is the ester (e.g. methyl or ethyl) of sarcosine, which is anester of N-methyl glycine.

Every possible permutation of ¹⁵N or ¹³C labeled glycine is commerciallyavailable. Likewise, other natural amino acids are commerciallyavailable with one or more incorporated heavy atom isotopes. Becauseglycine, and other amino acids, comprising one or more heavy atomisotopes are commercially available, these amino acids can be easilyincorporated into the procedure for the production of N-substitutedpiperazines. The amino acids comprising heavy atom isotopes can beN-protected using procedures well-known in peptide chemistry. Forexample, the amino acids can be N-protected with a9-fluorenylmethoxycarbonyl (Fmoc) group or a t-boc group. Furthermorethe amino acids comprising heavy atom isotopes can be N-alkylated andconverted to an ester of the amino acid using well-known procedures.Accordingly, heavy atom isotope containing starting materials for thepreparation of N-substituted piperazines, as described herein, areeither commercially available, or can be easily prepared fromcommercially available amino acids using no more than routineexperimentation.

According to the method, the two amino acids can be condensed to therebyproduce an N-protected peptide dimer as an ester. The N-protectedpeptide dimer ester can comprise one or more heavy atom isotopes via theincorporation of the one or more amino acids comprising one or moreheavy atom isotopes. The N-protected peptide dimer ester can compriseone heavy atom isotope, two heavy atom isotopes, three heavy atomisotopes, four heavy atom isotopes, five heavy atom isotopes or sixheavy atom isotopes. The N-protected peptide dimer ester can have thegeneral formula:

wherein Pg, R, Y and Z are previously defined.

According to the method, the N-protected peptide dimer ester can then becyclized to form a 6-membered cyclic dione. Cyclization proceeds byremoving the N-protecting group of the N-protected peptide dimer esterand driving the reaction of the deprotected amine with the ester group.The reaction can be carried out under basic conditions and can be heatedto speed production of the product. The product of the cyclization canhave the general formula:

wherein Y and Z are previously defined.

According to the method, the ketone groups of the cyclic dione can thenbe reduced to form the desired N-substituted piperazine comprising oneor more heavy atom isotopes. The reduction can be performed using areducing agent, such as lithium aluminum hydride (LAH) or Red-Al(Sigma-Aldrich). The product, in some embodiments being a volatile oil,can be optionally temporarily modified (e.g. protected) to aid inisolation. Because piperazine comprises two basic nitrogen atoms, theproduct can, in some embodiments, be isolated as a mono or bis-acidsalt. For example, the N-substituted piperazine comprising one or moreheavy atom isotopes can be isolated as a mono-TFA salt, a mono-HCl salt,a bis-TFA salt or a bis-HCl salt.

FIG. 1 illustrates the application of the aforementioned generalprocedure to the production of N-methyl piperazine. Examples 1-4describe the application of the illustrated procedure to the productionof three different N-methyl piperazines each comprising 1-3 heavy atomisotopes.

With reference to FIG. 1 and Examples 1-4, t-boc protected glycine (1)is condensed with sarcosine methyl ester (2) to thereby produce thedipeptide (3). The t-boc group is removed and the dipeptide is cyclizedto the cyclic dione (4). The ketone groups of the dione are then reducedto produce N-methyl piperazine. The N-methyl piperazine product caneither be transiently protected (5) or can be obtained directly from thereduction (6). The product can also be obtained as a salt (e.g. TFA salt(7) or HCl (8)) of an acid.

In summary, a wide variety of N-substituted piperazine compounds,unlabeled or labeled with one or more heavy atom isotopes, can beproduced by the aforementioned process. Consequently, the presentinvention contemplates all possible isotopically enriched N-substitutedpiperazine compound comprising one or more heavy atom isotopes of thegeneral formula:

including all possible salt forms thereof, wherein Y and Z arepreviously defined.II. Preparation of N-Substituted Piperazine Acetic Acids ComprisingHeavy Atom Isotopes

In some embodiments, this invention pertains to methods for theproduction of isotopically enriched N-substituted piperazine acetic aswell as the isotopically enriched N-substituted piperazine acetic acids.In some embodiments, an N-substituted piperazine can be reacted with ahalo acetic acid moiety comprising one or more heavy atom isotopes. Inthis context, halo refers to the halogens, chlorine, bromine and iodine.In still some other embodiments, an N-substituted piperazine comprisingone or more heavy atom isotopes can be reacted with a halo acetic acidmoiety. In some other embodiments, an N-substituted piperazinecomprising one or more heavy atom isotopes can be reacted with a haloacetic acid moiety comprising one or more heavy atom isotopes.Accordingly, the heavy atom isotopes found in the product N-substitutedpiperazine acetic acids can be introduced by way of the piperazine, byway of the halo acetic acid moiety or by way of both the piperazine andthe halo acetic acid moiety. As will be discussed in more detail below,¹⁸O can also be introduced into the carboxylic acid moiety of anN-substituted piperazine acetic acid by way of exchange with H₂ ¹⁸O.

Numerous light (by light we mean that the compound is not isotopicallyenriched with one or more heavy atom isotopes) N-substituted piperazines(e.g. N-methyl and N-ethyl piperazine) are commercially available.Furthermore, Section I above describes the preparation of N-substitutedpiperazine comprising one or more heavy atoms from commerciallyavailable amino acids. Both light and heavy (by heavy we mean that thecompound has been isotopically enriched with one or more heavy atomisotopes) N-substituted piperazine can be used to produce theN-substituted piperazine acetic acids comprising one or more heavy atomisotopes.

Numerous light and heavy halo acetic acid moieties are commerciallyavailable. The halo acetic acid moiety to be reacted with theN-substituted piperazine can be purchased as the carboxylic acid or asan ester of the carboxylic acid (e.g. the methyl ester, ethyl ester orphenyl ester). If only the carboxylic acid is available and the ester isdesired, the ester can be prepared using well-known esterificationmethods. If only the ester is available and the carboxylic acid isdesired, the ester can be hydrolyzed to produce the carboxylic acid.Either the carboxylic acid or the ester can be used in the alkylationreaction provided that an additional equivalent of base is required ifthe carboxylic acid is used. If the ester is used to perform thealkylation, the product ester can be hydrolyzed to produce theN-substituted piperazine acetic acid. General structures for thecarboxylic acid and the ester compounds that can be used to alkylateN-substituted piperazines are:

wherein Z and R are defined above. Hal is a halogen (Cl, Br or I) and Xis oxygen (O) or sulfur (S). In some embodiments, X is ¹⁶O or ¹⁸O. Oneor more of the atoms of the halo acetic acid compound can be a heavyatom isotope.

The alkylation of an N-substituted piperazine with a halo acetic acidmoiety proceeds under basic conditions. The base need only be strongenough to deprotonate piperazine but can be selected to notsubstantially react with the halo acetic acid moiety. In someembodiments, two or more equivalents of N-substituted piperazine can beused, as N-substituted piperazine is a base. If it is desirable to useonly one equivalent of N-substituted piperazine (for example, when theN-substituted piperazine is labeled with one or more heavy atom isotopesand is therefore valuable), other bases can be used. Suitable basesinclude, but are not limited to, hindered bases such as triethylamine(Et₃N) and diisopropylethyamine (DIEPA). Other suitable bases in sodiumcarbonate and potassium carbonate. Hindered bases are a good choicebecause they do not react substantially with the halo acetic acidmoiety.

A solid phase base, such as 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD)bound to polystyrene crosslinked with 2% DVB, Capacity (base): ˜2.6mmol/g (ss-TBD, Fluka, P/N 90603) can also be used (See FIG. 2B). Asolid phase base has the advantage that it is easily, and completely,removed from the product by filtration once the alkylation reaction hasbeen completed. Accordingly, the resulting product is not contaminatedwith salt of the base.

If the carboxylic acid is used to alkylate the N-substituted piperazine,the reaction can produce a product of the general formula:

or a salt thereof, wherein X, Y and Z have been previously defined. Oneor more atoms of the N-substituted piperazine acetic acid can be a heavyatom isotope.

If the ester is used to alkylate the N-substituted piperazine, thereaction will produce an ester of the general formula:

or a salt thereof, wherein R, X, Y and Z have been previously defined.One or more atoms of the N-substituted piperazine acetic acid ester canbe a heavy atom isotope. The N-substituted piperazine acetic acid estercan be converted to the N-substituted piperazine acetic acid of generalformula:

by hydrolysis of the ester. Depending on the state of protonation of theester, it may or may not be necessary to and base to aqueous solution toperform the hydrolysis because piperazine is basic (unless neutralizedby acid). Accordingly, base can be added as required to induce thehydrolysis of the ester to the carboxylic acid, but in some embodimentsit will not be required. Hydrolysis can also be performed under aqueousacidic conditions.

N-substituted piperazine acetic acid is zwitterionic. Because itcomprises a carboxylic acid group (or thio acid group) and two basicnitrogen atoms, it can exist in at least four different forms. It canexist completely deprotonated as its carboxylate anion. It can exist asits mono protonated zwitterion. It can exist as a monobasic salt (e.g.mono-TFA or mono-HCl salt). It can also exist as its dibasic salt (e.g.bis-TFA or bis-HCl salt). The state of protonation of the product is afunction of the conditions under which it was isolated. All protonationstates of N-substituted piperazine acetic acid are contemplated asembodiments of the present invention.

With reference to FIGS. 2A and 2B, as well as Examples 5 and 6,respectively, the production of two different isotopically enrichedN-methyl piperazine acetic acid compounds is described. In FIG. 2A andExample 5, two equivalents of commercially available unlabeled N-methylpiperazine is reacted with ethyl bromoacetate to produce a N-methylpiperazine acetic acid compound comprising two ¹³C atoms. BecauseN-methyl piperazine is basic, hydrolysis of the ethyl ester proceeded bymerely heating the compound in an aqueous solution.

With reference to FIG. 2B and Example 6, the starting piperazine is abis-TFA salt of ¹⁵N labeled N-methyl piperazine. Acid salts of thepiperazine base can be alkylated so long as sufficient base is added tothe reaction to deprotonate piperazine. In this example, the ethylbromoacetate is ¹³C labeled. Because both the piperazine and acetic acidreactants comprise heavy atom isotopes, a solid phase base was chosen sothat only one equivalent of each reactant was required to produce theproduct. As was observed with Example 5, hydrolysis of the ethyl esterproceeded by mere heating the compound in an aqueous solution.

In some other embodiments, the N-substituted piperazine acetic acid canbe assembled on a solid support. According to the method and withreference to FIG. 2C, the halo acetic acid moiety, as a carboxylic acid,can be reached with trityl chloride resin to thereby produce a supportbound halo acetic acid. The support bound halo acetic acid can then betreated with the desired N-substituted piperazine (e.g. N-methylpiperazine) under basic conditions to thereby produce the N-substitutedpiperazine acetic acid. Isotopically enriched N-methyl piperazine andhalo acetic acid moieties can be used, including ¹⁸O labeled compoundsalthough ¹⁸O labeling can involve special considerations and isdiscussed in more detail below.

In accordance with the aforementioned discussion, a heavy atom isotopecan be incorporated at virtually any position of the N-substitutedpiperazine acetic acid, including ¹⁸O incorporation that will bediscussed in more detail below. Consequently, the present inventioncontemplates all possible isotopically enriched N-substituted piperazineacetic acids comprising one or more heavy atom isotopes of the generalformula:

including all possible salt forms thereof.III. Incorporation of ¹⁸O Into N-Substituted Piperazine Acetic Acids

In some embodiments, this invention pertains to methods for theincorporation of ¹⁸O into N-substituted piperazine acetic acids as wellas to the ¹⁸O labeled N-substituted piperazine acetic acids themselves.In some embodiments, incorporation of ¹⁸O is not substantially differentas compared with the methods described for the preparation ofisotopically labeled N-substituted piperazine acetic acids in SectionII, above. In some other embodiments, incorporation of ¹⁸O issubstantially different and takes advantage of the very caveat thatcreates some concern about the methods previously discussed.

The caveat with respect to the preparation of ¹⁸O labeled N-substitutedpiperazine acetic acids lies with the exchange of ¹⁸O

¹⁶O that can occur between unlabeled water (H₂ ¹⁶O) and the ¹⁸O of aheavy carboxylic acid group. A carboxylic acid group is inherentlyacidic. Acid can catalyze the exchange of the oxygen atoms of acarboxylic acid group and water, such as residual water in a sample orwater used in a reaction (e.g. hydrolysis of an ester). Consequently,whenever ¹⁸O labeled N-substituted piperazine acetic acids were desired,one of two different synthetic routes was chosen.

In some embodiments, the ¹⁸O labeled N-substituted piperazine aceticacid was obtained by alkylation with an appropriately ¹⁸O labeled haloacetic acid moiety. The procedure is essentially as outlined in SectionII, above except that an acid labile ester of the halo acetic acid wasused in the alkylation reaction. In some embodiments, the halo aceticacid moiety comprised the formula:

wherein Hal is previously defined and R′ is an acid labile ester group,including but not limited to tert-butyldimethylsilyl or t-boc.

With reference to FIG. 3A and Example 8, the tert-butyldimethylsilyl(TBDMS) ester of (¹⁸O)₂ bromoacetic acid (14) was used in the alkylationreaction. This ester was prepared using ¹⁸O labeled bromoacetic acid(13), obtained as a custom order from Cambridge Isotope Laboratory,Inc., and TBDMS-CN. The TBDMS ester of N-methyl piperazine acetic acid(15) was the product of the alkylation with N-methyl piperazine. TheTBDMS ester was selected so that it could be converted to the acidchloride with, for example, oxalyl chloride thereby avoiding therequirement for any water and the possible exchange of ¹⁸O with ¹⁶O. Inthe presence of solid phase base (ss-TBD) and N-hydroxysuccinimide(NHS), the acid chloride was converted to the NHS ester (16). If thecarboxylic acid is desired, instead of the active ester, the TBDMS estercould be converted to the carboxylic acid by treatment with an anhydrosuch as TFA. Accordingly, aqueous treatment that might lead to ¹⁸O

¹⁶O exchange, can be avoided whether the active ester or the carboxylicacid is desired.

In some other embodiments, the alkylation to produce N-substitutedpiperazine acetic acid proceeded as described in Section II, above andthe ¹⁸O was later incorporated. With reference to FIG. 3B and Example 9,it was found that ¹⁸O could be incorporated into the carboxylic acidgroup of any N-substituted piperazine acetic acid by treatment of theN-substituted piperazine acetic acid with H₂ ¹⁸O under acidicconditions. For example and with reference to FIG. 3B, an isotopicallyenriched N-methyl piperazine acetic acid (17) lacking ¹⁸O, used toproduce the 114 labeling reagent, was treated with H₂ ¹⁸0 and either HClor TFA to thereby produce the TFA or HCl salt of the ¹⁸O isotopicallyenriched N-methyl piperazine acetic acid (18) and (19).

Furthermore, the isotopic purity of the product could be increased byrepeated cycles of treatment with H₂ ¹⁸O under acidic conditions. Thehigher the state of enrichment of the H₂ ¹⁸O, the fewer cycles requiredto produce highly ¹⁸O enriched N-substituted piperazine acetic acid.When H₂ ¹⁸O of 99% purity was used, the isotopic enrichment ofN-substituted piperazine acetic acid was typically 96% after two cycles.Because this exchange was performed under acidic conditions, the productwas easily isolated as the bis-acid salt of N-substituted piperazineacetic acid (e.g. the bis-TFA or bis-HCl salt).

Consequently, the present invention contemplates all possibleisotopically enriched N-substituted piperazine acetic acids comprisingone or more heavy atom isotopes of the general formula:

including all possible salt forms thereof.IV. Preparation of Various Active Esters of N-Substituted PiperazineAcetic Acid

In some embodiments, this invention pertains to methods for thepreparation of active esters of N-substituted piperazine acetic acid,including isotopically enriched versions thereof, as well as theN-substituted piperazine acetic acid esters themselves, and isotopicallyenriched versions thereof. The active ester can be any active ester. Insome embodiments, the active ester can be formed using an alcohol orthiol of the following formula:

wherein X is O or S, but preferably O. In some other embodiments, theactive ester can be formed using an alcohol or thiol of the followingformula:

wherein X is O or S, but preferably O.

In some embodiments, the active ester can be prepared through anintermediary imidazolide. According to this method, an N-substitutedpiperazine acetic acid ester, including isotopically enriched versionsthereof, can be converted to the imidazolide. The imidazolide soprepared can then be reacted with the alcohol of choice to therebyproduce the active ester of the selected alcohol.

With reference to FIG. 4A and Example 10, this procedure was used toprepare active esters of 2,2,2-trifluorethanol and1,1,1,3,3,3-hexafluoro-2-propanol. According to the figure and theexample, the phenyl ester of N-methyl piperazine acetic acid (20) wastreated with trimethyl silyl imidizole (TMS-imidizole) and sodiumphenoxide to form the imidazolide of N-methyl piperazine acetic acid(21). The imidazolide (21) was then reacted with either2,2,2-trifluorethanol or 1,1,1,3,3,3-hexafluoro-2-propanol to producethe desired active ester of N-methyl piperazine acetic acid (22) or(23), respectively as a bis-acid salt.

In some other embodiments, the active ester can be prepared byconversion of the N-substituted piperazine acetic acid, includingisotopically enriched versions thereof, to an acid chloride followed bysubsequent reaction of the acid chloride with the alcohol of choice tothereby produce the active ester of the selected alcohol.

With reference to FIG. 4B and Example 11, the preparation of the NHS andNHP esters of N-methyl piperazine acetic acid are illustrated using thisgeneral procedure. According to the figure and the example, N-methylpiperazine acetic acid is treated with oxalyl chloride to produce theacid chloride (24). The acid chloride is then treated with either of NHPor NHS and solid phase base to thereby produce the active ester (25) or(26), respectively as the free piperazine base (not as an acid salt).

FIG. 4B also illustrates the application of oxalyl chloride to theproduction of the pentafluorophenyl (Pfp) ester (27) wherein a solutionphase base (e.g. triethylamine) is used. The reaction proceeded wellwith the solution phase base but the hydrochloride salt of the baseproved difficult to remove. Application of the solid phase base avoidsthis caveat.

In still some other embodiments, the active ester can be prepared bytreatment of the N-substituted piperazine acetic acid, includingisotopically enriched versions thereof, with a trihalooacetate ester ofthe alcohol that is desired to form the active ester of theN-substituted piperazine acetic acid. In this context, halo refers tofluorine, chlorine, bromine and iodine but preferably to fluorine andchlorine. The trihalooacetate ester has the general formula:

wherein Hal refers to a halogen (fluorine, chlorine, bromine or iodine)and LG refers to the leaving group alcohol. The leaving group (LG) ofthe trihaloacetate esters can have the following general formula:

wherein X is O or S, but preferably O. Active esters of N-methylpiperazine acetic acid comprising these leaving groups (LG) weresuccessfully prepared using the identified trifluoroacetate esters(where X is O).

This procedure can be applied to the N-substituted piperazine aceticacids whether they are the acid salt or the zwitterion form. TheN-substituted piperazine acetic acids can be reacted with thetriholoacetate ester of the alcohol to thereby produce the active esterof the N-substituted piperazine acetic acid. A base that can deprotonatethe basic nitrogen atoms of piperazine ring can be added to the reactionas need to induce formation of the product when the starting material isan acid salt of N-substituted piperazine acetic acid. The active esterof the N-substituted piperazine acetic acid can itself be isolated asthe mono-acid salt or the di-acid salt. (e.g. the mono-TFA salt, themono HCl salt, the bis-TFA salt or the bis-HCl salt.). When thetrihalooacetate ester is reacted with an N-substituted piperazine aceticacid the product can be:

or a salt thereof, wherein X, Y and Z are previously defined. The groupLG is the leaving group of the active ester that is displaced by thereactive group of an analyte to be labeled; in essence the leaving groupis the alcohol used to form the active ester.

Certain trihaloacetate esters are commercially available. For example,the trifluoracetate esters of pentafluorphenol and 4-nitrophenol can bepurchased from commercial sources. However, the others can be obtainedby reacting the desired alcohol with trihaloacetic anhydride. Withreference to Table 1, below, the trifluoroacetate esters of Pcp, Dhbt,NHS, 3-NP and NHP were prepared by reacting the respective alcohol withtrifluoracetic anhydride. The general procedure for such reactions canbe found in Example 12. Other alcohols that can be used to producetrihaloacetate esters suitable for the formation of other active esterscan also be used.

FIG. 4C illustrates the production of the 114 and 115 labeling reagentsas the NHS ester. Accordingly, the procedure was successfully applied tothe production of isotopically enriched active esters of N-substitutedpiperazine acetic acids. These active ester reagents were produced asthe bis-HCl salts from the bis-HCl salts of the piperazine base.

FIG. 4D illustrates the production of numerous other active esters ofN-methyl piperazine acetic acid that were produced using this genericprocess. As will be appreciated by the ordinary practitioner, thisprocedure is generic and robust and can be applied to the production ofnumerous other active esters of a plethora of N-substituted piperazineacetic acid derivatives.

V. Isotope Incorporation Pathway for the Preparation of a Set ofIsobaric Labeling Reagents

FIG. 5A illustrates the general pathway taken to the production of a setof four isobaric labeling reagents identified as 114, 115, 116 and 117.These designations are based upon the “signature ion” each reagentproduces upon fragmentation in a mass spectrometer (FIG. 5B). The“signature ion” can be used to deconvolute information associated withdifferent samples in a multiplex assay as discussed in the Introduction.

The pathways illustrated in FIG. 5A utilize the procedures set forthabove for the production of N-substituted piperazine acetic acids, andactive esters thereof. In particular, suitable isotopically labeledglycines were used in the preparation of suitable isotopically labeledN-substituted piperazines (e.g. N-methyl piperazines). The labeled andunlabeled N-methyl piperazines can be treated with isotopically labeledbromoacetic acid derivatives, with or without subsequent ¹⁸O enrichmentto thereby produce the N-methyl piperazine acetic acid compounds ofdesired structure. These suitably labeled N-methyl piperazine aceticacid compounds were used as labeling reagents; in the present case byconversion to an active ester for coupling with analytes such aspeptides.

All four of the labeling reagents (114, 115, 116 and 117) were producedas NHS esters. All four reagents were used to label peptides, includingpeptides (analytes) obtained from digested protein. The set of reagents(two or more of them), were shown to be suitable for the multiplexanalysis, including proteome analysis, as described in copending andco-owned application Ser. No. 60/443,612, incorporated herein byreference.

For example, two or more samples containing digested peptides as theanalyte, each sample being labeled with one of the isobaric labelingreagents (114, 115, 116 or 117), were mixed to form a mixture that wasanalyzed in a tandem mass spectrometer. After the first MS analysis,selected ions, of a particular mass representing a mixture of fragmentions of the same analyte labeled with two or more different isobariclabels, were subjected to dissociative energy causing fragmentation ofthe selected ions. The selected ions, and the fragments thereof, werethen re-analyzed in the mass spectrometer wherein signature ions of theisobaric labeling reagents used to label the analytes, as well asdaughter ions of the analyte, were observed.

VI. State of Isotopic Enrichment

The various N-substituted piperazines, N-substituted piperazine aceticacids and active esters of N-substituted piperazine acetic acid can beprepared with starting materials of greater than 80 percent isotopicpurity of for each heavy atom isotope. The isotopic purity can begreater than 93 percent for each heavy atom isotope in some startingmaterials. In other starting materials the isotopic purity can begreater than 96 percent for each heavy atom isotope. In still otherstarting materials the isotopic purity can be greater than 98 percentfor each heavy atom isotope. When performing an ¹⁶O to ¹⁸O exchange, itwas possible to routinely obtain carboxylic acid groups of 96 or greaterpercent isotopic purity (per oxygen atom) of the heavy atom isotope.

Because, with the exception of ¹⁸O which can be exchanged with ¹⁶O incertain cases, the isotope purity and composition of starting materialswill translate directly into the isotopic purity of the products.Moreover, for ¹⁸O, it has been shown that isotopic purity of greaterthan 96 percent (per atom) can be achieved using the methods describedherein. Accordingly, in some embodiments, this invention pertains toN-substituted piperazines, N-substituted piperazine acetic acids and/oractive esters of N-substituted piperazine acetic acid having an isotopicpurity of at least 80 percent for each heavy atom isotope. In some otherembodiments, this invention pertains to N-substituted piperazines,N-substituted piperazine acetic acids and/or active esters ofN-substituted piperazine acetic acid having an isotopic purity of atleast 93 percent for each heavy atom isotope. In still some otherembodiments, this invention pertains to N-substituted piperazines,N-substituted piperazine acetic acids and/or active esters ofN-substituted piperazine acetic acid having an isotopic purity of atleast 96 percent for each heavy atom isotope. In yet some otherembodiments, this invention pertains to N-substituted piperazines,N-substituted piperazine acetic acids and/or active esters ofN-substituted piperazine acetic acid having an isotopic purity of atleast 98 percent for each heavy atom isotope.

The following examples are illustrative of the disclosed compositionsand methods, and are not intended to be limit the scope of theinvention. Without departing from the spirit and scope of the invention,various changes and modifications of the invention will be clear to oneskilled in the art and can be made to adapt the invention to varioususes and conditions. Thus, other embodiments are encompassed.

EXAMPLES

General Synthetic Notes: Unless otherwise stated, chemicals werepurchased from commercial sources and used as received. Unless otherwisestated, the following chemicals were purchased from Sigma-Aldrich.Trifluoroacetic anhydride (TFAA, P/N 106232), N-Hydroxysuccinimide (NHS,P/N 13067-2), tert-Butyl bromoacetate (P/N 124230), 4-Nitrophenyl (4-NP)trifluoroacetate (P/N N22657), Pentafluorophenyl (Pfp) trifluoroacetate(P/N 377074), tert-butyldimethylsilyl (TBDMS) cyanide (407852),1-(Trimethylsilyl)imidazole (P/N 153583), Phenyl bromoacetate (P/N404276), Pentachlorophenol (Pcp-OH, P/N P2604), 2,2,2-Trifluoroethanol(P/N 326747), 1,1,1,3,3,3-Hexafluoro-2-propanol (HFI-OH, P/N 105228),(3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (Dhbt-OH, P/N 327964), Oxalylchloride (P/N 320420), 1-Methylpiperazine (P/N 130001), Tetrahydrofuran(THF dry, P/N 186562). Dichloromethane (DCM dry, P/N 270997), 4 Mhydrochloric acid (HCl) solution in dioxane (P/N 345547), HCl (gas, P/N,295426), 3-Nitrophenol (3-NP-OH, P/N 163031)1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) bound to polystyrenecrosslinked with 2% DVB, Capacity (base): ˜2.6 mmol/g (ss-TBD, Fluka,P/N 90603), H₂ ¹⁸O (Isotec, 95 ¹⁸O atom % (P/N 329878) or 99% ¹⁸O atom %(P/N 487090)), Br¹³CH₂COOEt (Cambridge Isotope Laboratories (CIL), P/NCLM-1010-5), Br¹³CH₂ ¹³COOEt (CIL, P/N CLM-1011-1), BrCH₂C¹⁸O¹⁸OH(Isotec, P/N 597031). All moisture sensitive reactions were performedunder nitrogen or argon atmosphere.

Isotopically enriched starting materials were generally obtained fromeither Isotec (a Sigma-Aldrich company) or Cambridge IsotopeLaboratories (Andover, Mass.). Generally, the most highly enrichedstarting materials were obtained and used in the production of theisotopically enriched piperazine derivatives. However, the state ofisotopic enrichment of starting materials is a choice which the ordinarypractitioner will appreciate strikes a balance between the price of thestarting materials (wherein the higher the state of isotopic enrichment,the higher the price) and requirement for purity of the enrichedisotopes in the final product. Accordingly, the ordinary practitionerwill appreciate that the most practical method of synthesis ofisotopically enriched compounds may not always proceed through the mostcommon synthetic routes. Indeed, there may be two or more differentroutes to the different isotopic variants of the same compound. Thus,for some reactions and/or compounds described herein, various syntheticroutes have been undertaken and are therefore discussed below. Certainadvantages and caveats pertaining to these routes are also discussed.

I. Synthesis of Isotopically Labeled N-Methyl Piperazines

Note: Unlabeled N-methyl piperazine (a.k.a. 1-methyl piperazine) iscommercially available from a variety of sources. However, no source forany type of isotopically enriched N-methyl piperazine (as a stock item)could be found. It was determined however that suitably protectedglycine and sarcosine could be condensed, cyclized and the product (adiketone) could be reduced to thereby produce N-methyl piperazine (SeeFIG. 1). Furthermore, it was determined that all possible permutationsof ¹⁵N and ¹³C isotopically labeled glycine, as well as partiallyprotected versions thereof (e.g. t-boc protected amino acids), werecommercially available from sources such as Isotec or Cambridge IsotopeLaboratory, Inc. Accordingly, this appeared to be a promising route tovarious N-methyl piperazine compounds comprising one or more heavyatoms. Because appropriately protected (t-boc) isotopically enrichedglycines and suitably protected sarcosine can be purchased fromcommercial sources and because the protection of amino acids, such asglycine and sarcosine, are well-known, this discussion of the syntheticroute to N-methyl piperazine begins with the suitably protected aminoacids (FIG. 1).

Example 1 General Procedure for the Condensation of Sarcosine Ester andt-boc-Glycine (FIG. 1)

Note: Sarcosine is commercially available as either the methyl or theethyl ester. Either can be used in the condensation reaction.

To a round bottom flask (RBF) was added 1.1 equivalent (eq.) ofsarcosine ethyl ester (2) and 1 eq. of t-boc-glycine (1) (includingisotopically labeled t-boc-glycines for the production of variousisotopically labeled N-methyl piperazines). The solid was then dissolvedwith the addition of dichloromethane (DCM) (˜20 mL/g of t-boc-glycine).To this stirring solution was added 1.1 eq. of N-methyl morpholine (NMM)then 1.1 eq. of dicyclohexylcarbodiimide (DCC) in DCM. A precipitateformed within minutes. The reaction was stirred overnight. The reactionwas monitored by thin layer chromatography (TLC). If t-boc-glycine wasstill present, additional DCC in DCM was added. When the reaction wasdetermined to be complete, the solids were filtered off and the cake wasrinsed with DCM. The product containing solution was then evaporated todryness.

The product was purified by silica gel chromatography using a columnpacked in 50% ethyl acetate (EtOAc)/hexane. A small amount of the 50%EtOAc/hexane solution was used to dissolve/suspend the dried downproduct (not all will dissolve). This solution/slurry was loaded ontothe packed column. The column was eluted 50% EtOAc/hexane to obtain theminimally retained product. Product containing fractions were evaporatedto provide an oil, speckled oil, or flaky solid (materials that arehigher in heavy atom isotope content appeared to exhibit morecharacteristics of a solid).

TLC conditions: EtOAc (developed with ninhydrin and heat) Product (3)Rf~0.85 t-boc-glycine (1) Rf~0.3 (broad tailing) Sarcosine-OEt (2)Baseline NMM Faintly visible just above sarcosine

Example 2 General Procedure for the Synthesis of1-Methyl-2,5-Diketopiperazine (FIG. 1)

A solution of 1:1 trifluoroactetic acid (TFA):DCM containing 0.5% waterwas prepared. This solution was added to the column purified product (3)of the condensation reaction (˜10 mL/g starting material). The resultingsolution was stirred for 30 minutes and then the solvents removed byrotoevaporator. Ethanol (˜10 mL/g starting material) was then added tothe reaction flask and this solution was again striped to dryness. Theprocedure was repeated with toluene. The product was again dissolved inethanol in the reaction flask and anhydrous potassium carbonate (4 eq)was added. The solution bubbled vigorously for a short period followingthe addition of the potassium carbonate. A drop of the reaction mixturewas removed, diluted with water, and the pH of the solution wasdetermined. If the pH was below 8, more potassium carbonate was added.Once the pH was confirmed to be greater than 8, the reaction was allowedto reflux overnight. The warm reaction mixture was then passed through aplug of Celite to remove the excess salts. The cake was rinsed twicewith anhydrous ethanol. The filtrate was transferred to a larger flaskand stripped to dryness. The product foam was redissolved in 9:1 ethylacetate-methanol and passed through a plug of silica-gel. The silica-gelwas then washed with ˜4 column volumes of 9:1 ethyl acetate-methanol.All fractions were evaporated to dryness.

Notes and alternative procedures: Deprotection of the t-boc group withthe TFA/DCM/H₂O solution can be followed by TLC (ninhydrin/heat showsconversion of the spot at Rf 0.85 to a dark red-brown spot at origin).After deprotection, it is also acceptable to add methanol, concentrateand re-treat with methanol followed by a second concentration and dryingin vacuo to remove excess TFA.

In some reactions, concentrated ammonium hydroxide (large excess ˜60 mLper 16 mmol of starting material) was substituted for potassiumcarbonate. (When concentrated ammonium hydroxide was added to thereaction at room temperature, it generated a white insoluble materialand a slightly milky reaction mixture.) After the addition ofconcentrated ammonium hydroxide, the flask was sealed with septum toprevent loss of ammonia. Cyclization appeared to be complete afterovernight (12 hrs) reaction although in some cases heating to 60° C.over several hours was sufficient. The reaction was monitored by TLC(10% MeOH/DCM visualized with 10% phosphomolybdic acid (PMA) in MEOHwith heat). The product appeared as a blue spot at Rf 0.54. Since thedeblocked material (red-brown spot at origin) could not be visualizedwith PMA, another TLC was performed as a cross-reference usingninhydrin/heat.

When cyclization was deemed complete by TLC analysis, the mixture wasfiltered and the flask and solids were rinsed with DCM. The filtrate wasconcentrated and redissolved in 10% MeOH/DCM before chromatography. Thewhite waxy solids were partially insoluble in the 10% MeOH/DCM so thematerial was sonicated. Sonification successfully dissolved the mixturethat was then applied to the column. The first fraction eluted wasmainly the white waxy solid. The major fraction (the dione product (4))eluted next and was followed by another minor impurity (Rf 0.3). It wasobserved that in cases where incomplete TFA removal resulted information of ammonium triflate, this impurity co-eluted with the productand the secondary material. A second column could be used to completelypurify the desired product.

The melting points of the dione (4): 116,117: 138-139 for modelcompound: lit: 136-139 (J. Het. Chem 18, 423, 1981); 142-143 (J. Biol.Chem 61, 445, 1924).

TLC condition: 9:1 ethyl acetate-methanol (develop with phosphomolybdicacid and heat) Product Rf˜0.2 Alternative TLC condition: 10% MeOH/DCM;develop with heat Product (blue spot) Rf=0.54 1H-NMR data

NMR (D₆DMSO)—N1-CH₃, d 2.79, 2.80 3H; 3-CH₂ dd 3.94, 3.92, 3.59, 3.572H; 6-CH₂d 3.87, 3.86 2H; 4-NH b 8.11H.

NMR (D₂O)—N1-CH₃, d 3.00, 2.99 3H; 3-CH₂ d 4.08,4.08 2H, 6-CH₂ d 4.14,4.14 2H.

NMR (D₆ DMSO)—N1-CH₃, s 2.80 3H; 3-CH₂ s 3.76 2H; 6-CH₂ s 3.86 2H; 4-NHd 8.03,8.26 1H.

Example 3 General Procedure for the Synthesis of N-Methyl Piperazine(Route A; FIG. 1)

A saturated solution of sodium sulfate was prepared. Tetrahydrofuran(THF) (4 mL per mmol starting material based upon the material used inExample 2) was added to the diketopiperazine formed using the procedureof Example 2. The reaction flask was fit with a reflux condenser andthree equivalents of 1M LiAlH₄ in THF (LAH solution; it may be possibleto substitute Red-Al or other reducing reagent for LAH but this has notbeen attempted) was added to the solution through a dropping funnel.There was vigorous hydrogen evolution at the initiation of the additionbut this subsided as the addition continued. The reaction was heated toreflux for 4 hours. After the reaction was complete, the solution wascooled to room temperature and the remaining LAH was quenched with thevery slow addition of saturated aqueous sodium sulfate (¼ the volume ofthe LAH solution added). The reaction appeared as a gray suspension.

DCM was added to this suspension (½ volume of the THF) and the graygel-like solid was removed by filtration. The flask and filtered solidswere then thoroughly washed 2× with DCM (¼ volume of the THF). Thecombined organic solution (DCM/THF) was then dried with Na₂SO₄(solid-anhydrous) and filtered. (In some early experiments the N-methylpiperazine was isolated as an oil (free base and not as a TFA or HClsalt) but the product was determined to be a volatile oil and thereforenot be isolated in high yield).

Di-tert-butyl-dicarbonate (3 equivalents) was added to this solutionthat was stirred and vented overnight. TLC was used to monitor thereaction. Once complete, the solvent was removed by rotary evaporationto yield a liquid. This liquid is slightly volatile, so low vacuumevaporation of solvent is recommended (high vacuum conditions should beavoided). The product was dissolved in DCM and loaded onto a silica-gelcolumn packed with 8% methanol in ethyl acetate. Product was eluted withthe 8% methanol in ethyl acetate solution. Product containing fractionswere determined by TLC, pooled, and evaporated to a liquid. This liquidwas taken directly to the deprotection reaction. Note: the t-bocdeprotection was performed only as a means to isolate the crude N-methylpiperazine product but this requires subsequent deprotection.

TLC—N-methyl piperazine (develop with ninhydrin) 4:1:1Ethanol:Water:Ammonium hydroxide Product Rf=0.6 TLC—N¹-t-Boc-N²-methylpiperazine (develop with ninhydrin) 4:1 DCM-MeOH Product Rf=0.5

Deprotection:

A solution of 1:1 TFA, DCM with 0.5% water was prepared. This solutionwas added to the material isolated from the reduction, above (˜10 mL/gstarting material). The reaction was stirred for 30 minutes then thesolvent was removed with a rotoevaporator. Solvent evaporation wasterminated when no more solvent was observed to be collecting on thecondenser. TFA was added to the product residue (˜2 mL/g startingmaterial) to form a free flowing solution. The TFA solution wastransferred to a centrifuge tube and diethyl ether was added toprecipitate the product salt. The solution was mixed using a vortex. Thesolution was then centrifuged and the supernatant decanted to collectthe precipitate. The filtrate was then washed 1 time with ether byresuspending the product, vortexing, and re-centrifugation. Product wasdried under low vacuum to remove residual ether.

1H-NMR data:

NMR (D₂O)—N1-CH₃, d 3.02,3.03 3H; methylenes, broad triplet 3.3-3.9 8H

NMR (D₂O)—N1-CH₃, d 3.02,3.03 3H; methylenes, broad 3.40-3.85 8H

NMR (D₂O)—N1-CH, s 3.03 3H; methylenes, broad 3.50-3.75 8H

Example 4 General Procedure for the Synthesis of N-Methyl Piperazine(Route B; FIG. 1)

The product of the procedure of Example 2 was dissolved in anhydrous THF(5 mL per mmol SM) in a multi-neck RBF fitted with condenser, additionfunnel and argon (Ar) inlet. To this solution was added 3 equivalent ofthe LAH solution slowly through a dropping funnel at RT under Ar.Vigorous hydrogen evolution was observed at the beginning. Afteraddition, the cloudy solution was heated to reflux for 3 hours. TLC wasused to determine when the reaction was complete (disappearance ofstarting material (SM), 10% MeOH/DCM TLC developing solvent, PMA asvisualizer). After the reaction was complete, the solution was cooled toroom temperature and quenched with the very slow addition of saturatedaqueous sodium sulfate (¼ the volume of the LAH solution added). Whitegel-like solid solution was passed through a plug of Na₂SO₄ solid toremove H₂O. The filter cake was washed with THF several times (400 mLper gram SM) until TLC of the washing showed a little product. Then TFA(4 eq) was added to the THF solution (HCl in dioxane could also be addedif the HCl salt was desired). The color of the solution changed to lightbrown from pale yellow. The solution was concentrated on arotoevaporator under reduced pressure to yield brown oil. The lightbrown product was precipitated as bis-TFA salt by adding ether (42 mLper 1 gram SM) to yield of 80% N-methyl-piperazine. ¹H NMR (D₂O) wasused to confirm the desired product.

II. Alkylation of N-Methyl Piperazines to form N-Methyl PiperazineAcetic Acids

Note: FIG. 5A illustrates the pathway for the synthetic incorporation ofheavy atom isotopes into four isobaric labeling reagents referred toherein as 114, 115, 116 and 117. As can be seen from FIG. 5A, certain ofthe heavy atom isotopes can be incorporated by the choice of thecommercially available isotopically labeled glycine used in theproduction of the N-methyl piperazine. Certain other heavy atoms can beincorporated during the alkylation reaction based upon the choice of thecommercially available bromoacetic acid. In some cases, the ¹⁸O can beincorporated through an efficient exchange using ¹⁸O labeled water. Thelabeling reagents are designated 114, 115, 116 and 117 based upon themass of the fragment that forms a signature ion in the mass spectrometer(see: FIG. 5A and FIG. 5B) once the reagent has been fragmented by theapplication of dissociative energy.

With reference to FIG. 2A, scheme A is useful for producing the N-methylpiperazine acetic acid as a zwitterion and not as a salt (e.g. mono orbis TFA or HCl salt). With reference to FIG. 2B, scheme B is usefulsince it requires the use of only one equivalent of N-methyl piperazinefor the production of the N-methyl piperazine acetic acid therebyforeclosing the waste of the valuable isotopically labeled startingmaterial. With reference to FIG. 2C, scheme C is useful for alkylationsinvolving the isotopically labeled bromoacetic acid, particularly the¹⁸O labeled bromoacetic acid as it was expected to reduce the occurrenceof ¹⁸O scrambling (or exchange with ¹⁶O from residual water).

Example 5 Procedure for the Synthesis of Isotopically Labeled N-MethylPiperazine Acetic Acids (Scheme A; FIG. 2A)

To a stirring solution of 1.18 g (11.83 mmol) N-methyl piperazine in 15mL of toluene at room temperature was added 1 g (5.91 mmol) ofethylbromoacetate,1,2-¹³C dropwise, over a period of 15 minutes.Immediate formation of white solid was observed. The reaction mixturewas then heated in an oil bath at 90° C. for 4 hr. After cooling themixture to room temperature, the off-white solid was removed byfiltration, and washed with 25 mL of toluene. The combined filtrate andwashings was then concentrated in a rotoevaporator, and the residue wasdried under high vacuum for 5 hours to yield 1.10 g (quantitative) ofethyl ester of piperazine acetic acid-1,2-¹³C (9) as an off-white oil.The crude product (9) was analyzed by MS and ¹H-NMR, and was directlyused for the next step without further purification. MS (ESI, m/z):189.16 (M+1), ¹H-NMR (DMSOd₆) δ 4.2 (m, 2H), 3.4 (d, 1H, J=7Hz), 3.05(d,1H, J=7 Hz)), 2.4-2.7 (b, 8H), 2.3 (s, 3H), 1.25 (t, 3H).

A solution of ethyl ester of N-methyl piperazine acetic acid (9) (1.1 g,mmol), prepared as described above, in water (20 mL) was refluxed for 24hr. The reaction was monitored by MS analysis. After 24 hr, the reactionmixture was concentrated in a rotoevaporator to afford white solidproduct, which was triturated with acetone (10 mL) overnight. Theproduct was then separated by filtration and dried under high vacuumovernight at 45° C. in a vacuum oven, to yield 780 mg of N-methylpiperazine acetic acid, 1,2-¹³C (10), as a white powdery solid. 300 mgof the product was further purified by sublimation (1 mm/Hg, 110-120°C.) to yield 270 mg of white solid. MS (ESI, m/z); 161 (M+1), ¹H-NMR(DMSOd₆) δ 3.3 (d, 1H, J=7Hz), 2.95(d, 1H, J=7Hz), 2.55-2.75 (b,4H),2.3-2.45 (b,4H), 2.18 (s, 3H)

Notes: This procedure utilizes unlabeled N-methyl piperazine. Thisprocedure is useful for producing the zwitterion of N-methyl piperazineacetic acid.

The product can also be isolated as the mono or bis-HCl or mono orbis-TFA salt by treatment with the appropriate acid prior to orsubsequent to its isolation as described above.

Example 6 Procedure for the Synthesis of Isotopically Labeled N-MethylPiperazine Acetic Acids (Scheme B; FIG. 2B)

To a slurry of 200 mg (1.14 mmol) of N-methylpiperazine-¹⁵N.2HCl (the2TFA salt can also be used) in methanol (MeOH, 14 mL), was added 1.76 g(4.59 mmol) of ss-TBD, with aloading of 2.6 mmol/g, followed by CH₂Cl₂(6 mL). The mixture was then sonicated for 15 minutes and was thencooled in an ice bath under an argon atmosphere. To this vigorouslystirred slurry, a solution of 193 mg (1.14 mmol) ofethylbromoacetate-2-¹³C in acetonitrile (3 mL) was added dropwise usinga syringe pump (maintaining a rate of 2 mL/hr). After completion of theaddition, the ice bath was removed and the mixture was continuedstirring at room temperature overnight (18 hr). The mixture was thenfiltered through a sintered funnel, and the solid was washed severaltimes with MeOH (4×10 mL). The combined filtrate and washings were thenconcentrated in a rotoevaporator, and the residue was kept under highvacuum to yield 111 mg (51%) of the ethyl ester of the N-methylpiperazine acetic acid (11) as an off white solid. This crude productwas directly used for the next step without further purification. MS(ESI, m/z) 189 (M+1). ¹H-NMR (DMSOd₆) δ 4.05 (q, 2H), 3.3(s, 1H), 3.0(s, 1H), 2.4-2.5 (b, 4H), 2.2-2.4(b, 4H), 2.1 (s, 3H), 1.15 (t, 3H).

The product was hydrolyzed in the manner described in Scheme A, above.The following analytical data was obtained for the product.

MS (ESI, m/z) 161 (M+1). ¹H-NMR (DMSOd₆) δ 3.35(s, 1H), 3.05 (s, 1H),2.65-2.8(b, 4H), 2.5-2.65(b, 4H), 2.35 (s, 3H),

Without substantial variation, above general procedure was applied toother isotopically labeled N-methyl piperazines to produce variousisotopically labeled N-methyl piperazine acetic acid derivatives.

The product can also be isolated as the bis-HCl or bis-TFA salt bytreatment with the appropriate acid prior to or subsequent to itsisolation as described above.

Example 7 General Procedure for the Synthesis of Isotopically LabeledN-Methyl Piperazine Acetic Acids (Scheme C; FIG. 2C)

To a solution of bromoacetic acid (715 mg, 5 mmol) in DCM (15 mL) wasadded 700 mg of trityl-Cl resin (1 mmol, 1.45 mmol/g) followed bydiisopropylethylamine (DIPEA) (1.79 mL, 10 mmol). This solution wasmixed at room temperature for 1 hour. The resin was then filtered andwashed with dichloromethane (3×4 mL) followed by a wash with a solutionof dichloromethane-methanol-DIPEA (17:2:3,5 mL) and finally a wash withdichloromethane (3×4 mL).

The resin was then treated with a solution of N-methyl piperazine (N-MP)(0.57 mL, 5 mmol) in DMF (5mL) for 30 minutes and then washed with DMFand dichloromethane (3×4 mL each). The N-MPA so generated on resin wascleaved with a 25% solution of TFA in dichloromethane (10 mL for 5 min))and resin was washed with the same solution (2×5 mL). After evaporationof TFA, the product was precipitated and washed with ether (388 mg, 99%yield, bis TFA salt). The product was identified by NMR (matched withliterature) and with ES-MS (Calculated MH⁺=159.11, found 159.14).

Without substantial variation, the above general procedure was appliedto other isotopically labeled N-methyl piperazines to produce variousisotopically labeled N-methyl piperazine acetic acid derivatives.

The product could also be isolated as its bis-HCl salt if HCl was usedto cleave the product from the support rather than TFA. Other acidscould also be used for the cleavage reaction and product would be thesalt of the acid used.

III. Methods for the Incorporation of ¹⁸O into N-Methyl PiperazineAcetic Acids

Note: In the initial experiments, incorporation of ¹⁸O into the N-methylpiperazine acetic acid was attempted as illustrated in FIG. 3A using ¹⁸Olabeled bromoacetic acid (custom synthesized by CIL). Caveats to thisapproach include the possibility that in subsequent reactions, the ¹⁸Ocan exchange with ¹⁶O from residual water or can otherwise exchange with¹⁶O from other reagents during the esterification process. The morerecently applied synthetic procedure is illustrated in FIG. 3B andcapitalizes on the ¹⁶O

¹⁸O exchange reaction, using H₂ ¹⁸O to drive the equilibrium reaction toformation of the desired heavy version of the N-methyl piperazine aceticacid. Though both schemes have been shown to work, Scheme B currentlysupports the production of the most highly ¹⁸O enriched products.

Example 8 General Procedure for the Synthesis of ¹⁸O IsotopicallyLabeled N-Methyl Piperazine Acetic Acids, including Conversion to theActive Ester (Scheme A; FIG. 3A)

To a solution of TBDMS-CN (172 mg, 1.190 mmol)) in DCM (0.575 mL) wasadded ¹⁸O labeled bromoacetic acid (13) (170 mg, 1.189 mmol) under anargon atmosphere and the solution was heated to 80 ° C. for 20 minutesand then cooled to room temperature. The product (14) was isolated as anoil (254 mg, 85% yield). ¹H NMR (CDCl₃) δ 3.58 (2H, —CH ₂—), 0.955 (9H,(CH ₃)₃—Si), 0.30 (6H, CH₃Si).

A solution of BrCH₂C¹⁸O₂-TBDMS (14) (254 mg, 1 mmol) in DCM (2.5 mL) wasadded (34 μL/min) to an argon flushed flask containing N-MP (110 μL, 1mmol), TBD resin (576 mg, 1.5 mmol, 2.6 mmol/g) and DCM at 0 ° C. Afterthe addition was complete the reaction continued for 1 h at RT and thenthe resin was filtered and washed with DCM. Combined filtrate wasconcentrated by rotary evaporation to obtain 118 mg (42% yield) of anoil (15).

Note: Because of the potential for ¹⁸O

p¹⁶O exchange during the esterification, the N-methyl piperazine aceticacid prepared by this route was not converted to the active ester usingthe trifluoracetate procedure described in Section VI, below. Instead itwas converted using oxalyl chloride and NHS as described below.

To a solution of ¹⁸O containing TBDMS ester of N-MPA (15) as obtainedabove (118 mg, 0.427 mmol) in DCM (5 mL) was added a solution of oxalylchloride (0.427 mL, 0.854 mmol, 2 M solution in dichloromethane) at roomtemperature. The reaction allowed to continued for 1 hour when an offwhite slurry formed. Solvent and excess reagent were removed from thereaction mixture. A solution of NHS (50 mg, 0.427 mmol) in dry THF (1.4mL) was added to the resulting solid followed by 5 mL ofdichloromethane, 4 mL of THF and 246 mg of ss-TBD resin (0.640 mmol, 2.6mmol/g). The mixture was sonicated and mixed for 20 minutes, after whichthe resin was filtered and washed with 5 mL of dry dichloromethane. Tothe filtrate so obtained was added 2 mL of 4.0 M solution of HCl indioxane and the precipitate (16) was washed with dry THF (5 mL×2) andhexanes (5 mL) and dried under vacuum (10 mg, 7% yield). ES-MS (directinfusion in i-propanol) shows isotopic purity to be around 74% at thisstage.

Example 9 General Procedure for the Synthesis of ¹⁸O IsotopicallyLabeled N-Methyl Piperazine Acetic Acids (Scheme B; FIG. 3B)

200 mg (1.24 mmol) of N-methyl piperazine acetic acid 1,2-¹³C (17) wasweighed out in a 5 mL plastic vial flushed with argon. The vial was thentransferred into a glove box and 2.5 mL of ¹⁸O-water (>99% ¹⁸O) wasadded. The vial was then fitted with a silicone septum, and a low streamof HCl gas was then passed through the solution using a long needleafter venting the septum with an open needle. When the solution hadwarmed (˜2 min), the HCl passage was stopped, and the septum wasreplaced with a screw-cap. The vial was then heated at 80° C. in aheating block for 18 hr. An aliquot was analyzed by MS and ¹⁸O puritywas calculated as 93%. The reaction mixture was then concentrated todryness in a speedvac, and the residue was subjected to a second cycleof ¹⁸O-exchange as described above. By MS analysis the ¹⁸O purity afterthe second cycle was determined as 96%. The mixture was then evaporatedto dryness in a speedvac, and traces of water were removed byco-evaporataion with toluene (1 mL×2 ). 220 mg of N-methyl piperazineacetic acid-1,2-¹³C—¹⁸O_(2.)2HCl (18) was obtained. MS (ESI, m/z), 165(M+1)

Note: The product was used without further purification in theproduction of active ester of the N-methyl piperazine acetic acid. Thebis-TFA salt was also produced using the above-described procedurewherein TFA was substituted for HCl.

IV. Preparation of the Active Esters of the N-Methyl Piperazine AceticAcids

Note: Several methods were employed for the production of active estersof N-methyl piperazine acetic acid. The procedure illustrated by SchemeA (FIG. 4A) worked well for the production of the fluoroalcohol estersof N-methyl piperazine (See: FIG. 4C and 4D). The procedure illustratedby Scheme B (FIG. 4B) produced various active esters of N-methylpiperazine but unless the solid phase base was used (e.g. ss-TBD), thehydrochloride salt of solution phase base was difficult to remove. Theprocedure illustrated by Scheme C (FIGS. 4C and 4D) proved to be themost generally applicable route to the production of active esters ofN-methyl piperazine.

Example 10 Synthesis of Active Esters of N-methyl Piperazine Acetic AcidVia Imidazolide Formation (Scheme A, FIG. 4A)

To a solution of N-methyl piperazine phenyl ester (20) (100 mg, 0.426mmol) and sodium phenoxide (1 mg, 9 μmol) in THF (5 mL) was addedTMS-imidazole (69 μL, 0.468 mmol). The solution was mixed for 20 minutesto generate the imidazolide (21). CF₃CH₂OH (80 μL, 0.213 mmol) was thenadded to the light yellow solution so obtained. The solution was mixedfor another 30 minutes when TLC indicated clean formation of product(R_(f)=0.6, 4:1 DCM-MeOH). The reaction was then diluted to 15 mL withEtOAc and the product (22) was precipitated by addition of HCl solutionin dioxane (4 M, 2mL). After washing with THF (2×15 mL) product wasisolated as white solid. NMR of the solid indicated a 1:1 mixture ofproduct and imidazole (as HCl salt). Calculated MH⁺=241.13,found=241.12.

1,1,1,3,3,3-Hexafluoro-2-propanol ester (23) was isolated using thegeneral procedure set forth above provided however that (CF₃)₂CHOH wassubstituted for CF₃CH₂OH. The following analytical data was obtained forthis product. (R_(f)=0.37, 9:1 DCM-MeOH). Calculated MH⁺=309.11 ,found=309.11.

Note: N-methyl piperazine phenyl ester was prepared by the alkylationprocedures described above (See FIGS. 2A and 2B) wherein phenylbromoacetate is substituted for ethyl bromoacetate.

Example 11 Synthesis of Active Esters of N-Methyl Piperazine Acetic AcidVia Oxalyl Chloride (Scheme B, FIG. 4B)

To a suspension of N-methyl piperazine acetic acid (N-MPAA) (79 mg, 0.5mmol) in DCM (25 mL) was added a solution of oxalyl chloride (4 mL, 0.8mmol, 2.0 M solution in DCM) over 10 minute at room temperature. Afteranother 30 minutes of reaction, solvent and excess reagent were removedunder reduced pressure to give a white solid (24). A solution of NHS (57mg, 0.5 mmol) in DCM (25 mL) was added to the solid followed by ss-TBD(390 mg, 1 mmol, 2.6 mmol/g). The resulting solution was sonicated for 5minute when all solid dissolved. The ss-TBD resin was removed byfiltration and solvent was evaporated to yield a white foam (97% yield).Product was characterized by ES-MS as before.

Synthesis of Active Esters of N-Methyl Piperazine Acetic Acid ViaTrifluoroacetate Esters (Scheme C, FIGS. 4C and 4D)

Note: Conversion of the N-methyl piperazine acetic acids (N-MPAAs) totheir active -esters-via-the trifluoracetate ester is typically atwo-step process. Except for the rare case where the reagent iscommercially available (See: Table 1), the first step involves thepreparation of a reagent for esterifying the acetic acid. The secondstep involves reacting the esterifying reagent with the N-methylpiperazine acetic acid to produce the active ester. Various activeesters were produced and tested for the aqueous labeling of peptides.Though the NHS ester proved to be quite useful for this application,other esters may prove useful in other applications. Nevertheless, thismethod of producing the active esters proved to be quite robust andgenerally applicable across a wide variety of compounds. FIG. 4Billustrates 7 different active esters that were produced using the samegeneric procedure.

Example 12 Synthesis of N-Hydroxysuccinimide Trifluoroacetate^(10,11)and other Trifluoroacetate Esters

Trifluoroacetic anhydride (4.9 mL, 4×8.68 mmol (2.5-4 equivalents istypically used) was added to N-hydroxysuccinimide (NHS) (1 g, 8.69 mmol)and stirred under argon for 1-2 h to produce a homogeneous reactionmixture. Excess reagent and by-product CF₃COOH were removed underreduced pressure (rotary evaporation). The product was obtained as whitesolid in quantitative yield. The solid was dried under high vacuum for3-4 h and stored under argon (Ar) or nitrogen (N₂) gas.

With reference to FIG. 4D and Table 1, the trifluoroacetate ester ofpentafluorophenol (Pfp) and 4-nitrophenol (4-NP) were commerciallyavailable. The remaining trifluoroacetate esters were synthesized usingthe above-described generic procedure provided however that the reactiontime and temperature were varied. Furthermore, in some cases theproducts were isolated by distillation. Yields of the trifluoroacetateesters were good and in some cases near quantitative. The specificconditions used are set forth in Table 1, below.

TABLE 1 pK_(a) 4.68

Pcp 5.50

Pfp 7.23

4-NP 7.78

Dhbt 7.80

NHS 8.33

3-NP 9.38

NHP

Example 13 General Method for the Preparation of Active Esters ofN-Substituted Piperazine Acetic Acid from Trifluoroacetate Esters

A solution of the trifluoroacetate in THF (0.58 M, 1.2 equiv) was addedto a solid sample of N-methyl piperazine acetic acid and mixed in avortex or shaker until a homogeneous solution was obtained. The reactionof the carboxylic acid with the trifluoroacetate ester was generallycomplete within 30 min for all cases except N-hydroypyrrolidinone (NHP,18 h). The progress of conversion to the active ester was monitored byES-MS. The amount of product and any starting material (N-MPA) could bedetermined by direct infusion of a sample of the reaction (in ethanol)into the ES-MS. In some cases the active ester product was precipitatedas dihydrochloride salt by the addition of a solution by addition of HClsolution in dioxane (4 M, 50% volume of the reaction) followed bywashing with THF, ethyl acetate and hexanes. In other cases the productwas isolated from the reaction as the mono TFA salt. Addition of TFAcould be performed if the bis-TFA salt was desired.

Dhbt ester, Calculated MH⁺ = 304.14 Found = 304.20 NHP ester, CalculatedMH⁺ = 242.15 Found = 242.20 4-NP ester, Calculated MH⁺ = 280.13 Found =280.20

¹H NMR (400 MHz, CDCl₃) d 8.20 (d, 2H, J=9.2 Hz, aromatic protons), 7.25(d, 2H, J=9.2 Hz, aromatic protons), 3.69-3.40 (broad, 2H, ringprotons), 3.57 (s, 2H, —CH ₂—CO—), 3.15-2.90 (broad, 6H, ring protons),2.78 (s, 3H, —CH₃).

Pfp ester, Calculated MH⁺ = 325.10 Found = 325.10 Pcp ester, CalculatedMH⁺ = 404.95 Found = 405.90 3-NP ester, Calculated MH⁺ = 280.13 Found =280.20 NHS ester, Calculated MH⁺ = 256.13 Found = 256.10

Example 14 Synthesis of the NHS-ester of N-methyl piperazine aceticacid-1,2-¹³C—¹⁸O₂, 2.HCl (the 114 labeling reagent)

To a slurry of N-methyl piperazine acetic acid-1,2-¹³C, ¹⁸O, 2.HCl (28)(60 mg, 0.25 mmol) in THF (1.8 mL), was added DIPEA (98 mg, 0.76 mmol)under argon. The mixture was vortexed for 5 min, and thetrifluoroacetate of N-hydroxysuccinimide (160 mg, 0.76 mmol) was added.After sonicating for 10 minutes, the reaction mixture was stirred atroom temperature for 4 hours, followed by a centrifugation to remove anyundissolved material. The supernatant was decanted then diluted with THF(3 mL) and added slowly to a 4M solution of HCl in dioxane (1.8 mL). Theprecipitated HCl salt of the NHS-ester was separated by centrifugation,and washed with THF (3mL×4), dried under high vacuum to yield 62 mg(74%) of the NHS ester (30) as an off-white solid.

MS (ESI, m/z) 261 (M+1), ¹H-NMR (DMSOd₆) δ 4.05 (d, 1H, J=7Hz), 3.7 (d,1H, J=7Hz), 3.3-3.45 (b, 2H), 2.95-3.1 (b, 2H), 2.85(s, 3H), 2.75 (m,4H).

With the exception of using a different isotopically enriched N-methylpiperazine acetic acid, the above describe procedure was followed forthe production of the 115 labeling reagent (31). The analytical data forthe product (31) is as follows.

MS (ESI, m/z) 261 (M+1). ¹H-NMR (DMSOd6) δ 4.05(s, 1H), 3.7 (s, 1H),3.3-3.4 (b, 2H), 3.1-2.95(b, 4H), 2.85 (s, 3H), 2.75-2.80 (b, 1H), 2.7(m, 4H).

Notes: The trifluroacetate ester reagent can be reacted with thezwitterion of N-methyl piperazine acetic acid and well as with a monosalt or bis salt (e.g. mono-HCl salt, mono-TFA salt, bis-HCl salt orbis-TFA salt) of the N-methyl piperazine acetic acid. When the bis-HClsalt was used a base such as diisoproplyethylamine (DIPEA) was added toneutralize the acid. The transesterification reaction did howeverapparently proceed with the bis-TFA salt of N-methyl piperazine aceticacid without the addition of base.

The examples set forth above are for illustrative purposes only andshould not be viewed as a limitation on the scope of the invention.

REFERENCES

-   1 (a) Finkelstein, J. A.; Kruse, L. I.; Leonard, T. B. Dopamine    Beta-hydroxylase Inhibitors EP 0261804-A1, 1987 (b) Rautio, J.;    Nevalainen, T.; Taipale, H.; Vepsalainen, J.; Gynther, J.; Laine,    K.; Jarvinen, T. Synthesis and in vitro Evaluation of Novel    Morpholinyl- and m-Methylpiperazinylacyloxyalkyl Prodrugs of    2-(6-Methoxy-2-naphthyl)propionic acid (Naproxen) for Topical Drug    Delivery. J. Med. Chem. 2000, 43, 1489-1494.-   2 Nudelman, A.; McCaully, R. J.; Bell, S. C. Preparation of    1,4Benzodiazepines. American Home Products Corp. US3860581. Jan. 14,    1975.-   3 Wissner, A.; Grudzinskas, C. V. Reaction of    tert-Butyldimethylsilyl Esters with Oxalyl    Chloride-Dimethylformamide: Preparation of Carboxalic Acid Chlorides    under Neutral Conditions. J. Org. Chem. 1978, 43, 3972-3974.-   4 Heyes, M. P.; Markey, S. P. (180)-Quinolinic acid: its    esterification without back exchange for use as internal standard in    the quantification of brain and CSF quinolinic acid. Biomedical &    Environmental Mass Spectrometry, 1988, 15, 291-293.-   5 Biswas, A.; Miller, M. J. Rearrangement of    N-(p-Toluenesulfonyloxy)-2-Pyrrolidinone. Heterocycles, 1987, 26,    2849-2851.-   6 (a) Stacey, M.; Bourne, E. J.; Tatlow, J. C.; Tedder, J. M. A    General Method of Esterification using Trifluoroacetic anhydride.    Nature 1949, 164, 705. (b) Sakakibara, S. Nukai, N. A New Reagent    for the p-Nitrophenylation of Amino Acids. Bull. Chem. Soc. Jpn.    1964, 37, 1231.-   7 Krusic, P. J.; Chen, K. S.; Meakin, P.; Kochi, J. K. Electron Spin    Resonance Studies of Fluoroalkyl Radicals in Solution. III.    Photolysis of Perfluoroketones and Adduct Formation. J. Phys. Chem.    1974, 78, 2036-2047.-   8 Bates, G. S.; Diakur, J.; Masamune, S. Selective and Direct    Activation of O-esters. Conversion of Phenyl and    2,2,2-Trifluoroethyl Esters into Acyl Imidazolides. Tetrahedron    Letters, 1976, 49, 4423-4426.-   9 Bishop, B. F. New Antiparasitic Agents Related to the Milbemycins    and Avermectins. Pfizer Ltd. WO9415944, 21 Jul. 1994.-   10 Rao, T. Sudhakar; Nampalli, Satyam; Sekher, Padmanabhan; Kumar,    Shiv. TFA-NHS as Bifunctional Protecting Agent: Simultaneous    Protection and Activation of Amino Carboxylic Acids. Tetrahedron    Letters 2002, 43, 7793-7795.-   11 Sakakibara, Shumpei; Inukai, Noriyoshi. Trifluoroacetate Method    of Peptide Synthesis. I. The Synthesis and Use of Trifluoroacetate    Reagents. Bulletin of the Chemical Society of Japan, 1965, 38,    1979-1984.

1. An isotopically enriched N-substituted piperazine compound of theformula:

or a salt thereof wherein; Y is a straight chain or branched C1-C6 alkylgroup or a straight chain or branched C1-C6 alkyl ether group whereinthe carbon atoms of the alkyl group or alkyl ether group are eachindependently optionally substituted with deuterium or fluorine atoms;and each Z is independently hydrogen, fluorine, chlorine, bromine,iodine, an amino acid side chain, a straight chain or branched C1-C6alkyl group that may optionally contain a substituted or unsubstitutedaryl group wherein the carbon atoms of the alkyl and aryl groups areeach independently optionally substituted with fluorine atoms, astraight chain or branched C1-C6 alkyl ether group that may optionallycontain a substituted or unsubstituted aryl group wherein the carbonatoms of the alkyl and aryl groups are each independently optionallysubstituted with fluorine atoms or a straight chain or branched C1-C6alkoxy group that may optionally contain a substituted or unsubstitutedaryl group wherein the carbon atoms of the alkoxy and aryl groups areeach independently optionally substituted with fluorine atoms; whereinthe N-methyl piperazine is isotopically enriched with one or more ¹³Cand/or ¹⁵N atoms.
 2. The compound of claim 1, wherein the N-substitutedpiperazine is isotopically enriched with two or more atoms of ¹³C and/or¹⁵N.
 3. The compound of claim 1, wherein the N-substituted piperazine isisotopically enriched with three or more atoms of ¹³C and/or ¹⁵N.
 4. Thecompound of claim 1, wherein the N-substituted piperazine isisotopically enriched with four or more atoms of ¹³C and/or ¹⁵N.
 5. Thecompound of claim 1, wherein each Z is independently hydrogen, fluorine,chlorine, bromine or iodine.
 6. The compound of claim 1, wherein each Zis independently hydrogen, methyl or methoxy.
 7. The compound of claim1, wherein Y is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl or tert-butyl.
 8. The compound of claim 1, wherein eachnitrogen atom of the piperazine ring is independently ¹⁴N or ¹⁵N.
 9. Thecompound of claim 1 of the formula:

or salt of any of the fore going.
 10. The compound of claim 9, whereinthe compound is a mono-TFA salt, a mono-HCl salt, a bis-TFA salt or abis-HCl salt.
 11. The compound of claim 9, wherein each incorporatedheavy atom isotope is present in at least 80 percent isotopic purity.12. The compound of claim 9, wherein each incorporated heavy atomisotope is present in at least 93 percent isotopic purity.
 13. Thecompound of claim 9, wherein each incorporated heavy atom isotope ispresent in at least 96 percent isotopic purity.
 14. The compound ofclaim 1, wherein the N-substituted piperazine is a mono-TFA salt, amono-HCl salt, a bis-HCl salt or a bis-TFA salt.
 15. The compound ofclaim 1, wherein each incorporated heavy atom isotope is present in atleast 80 percent isotopic purity.
 16. The compound of claim 1, whereineach incorporated heavy atom isotope is present in at least 93 percentisotopic purity.
 17. The compound of claim 1, wherein each incorporatedheavy atom isotope is present in at least 96 percent isotopic purity.18. An isotopically enriched N-substituted piperazine compound of theformula:

or a salt thereof wherein; Y is a straight chain or branched C1-C6 alkylgroup or a straight chain or branched C1-C6 alkyl ether group whereinthe carbon atoms of the alkyl group or alkyl ether group are eachindependently optionally substituted with deuterium or fluorine atoms;and each Z is independently hydrogen, fluorine, chlorine, bromine,iodine, an amino acid side chain or a straight chain or branched C1-C6alkyl group that may optionally contain a substituted or unsubstitutedaryl group wherein the carbon atoms of the alkyl and aryl groups areeach independently optionally substituted with fluorine atoms; whereinthe N-substituted piperazine is isotopically enriched with one or more¹³C atoms and/or ¹⁵N atoms.
 19. The compound of claim 18, wherein each Zis hydrogen.
 20. An isotopically enriched N-substituted piperazinecompound of the formula:

or a salt thereof, wherein; Y is a straight chain or branched C1-C6alkyl group or a straight chain or branched CI-C6 alkyl ether groupwherein the carbon atoms of the alkyl group or alkyl ether group areeach independently optionally substituted with deuterium or fluorineatoms; and each Z is independently hydrogen, fluorine, chlorine,bromine, iodine, an amino acid side chain or a straight chain orbranched C1-C6 alkyl group that may optionally contain a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups are each independently optionally substituted with fluorineatoms; and wherein the N-substituted piperazine is isotopically enrichedwith one or more ¹³C atoms and/or ¹⁵N atoms.
 21. An isotopicallyenriched N-substituted piperazine compound of the formula:

or a salt thereof, wherein; Y is a straight chain or branched C1-C6alkyl group or a straight chain or branched C1-C6 alkyl ether group; andeach Z is independently hydrogen, fluorine, chlorine, bromine, iodine,an amino acid side chain, a straight chain or branched C1-C6 alkylgroup, a straight chain or branched C1-C6 alkyl ether group or astraight chain or branched C1-C6 alkoxy group; and wherein theN-substituted piperazine is isotopically enriched with one or more ¹³Catoms and/or ¹⁵N atoms.
 22. An isotopically enriched N-substitutedpiperazine compound of the formula:

or a salt thereof, wherein the N-substituted piperazine is isotopicallyenriched with one or more ¹³C atoms and/or ¹⁵N atoms.